This invention relates to a polypeptide with antifungal, anti-Oomycete and plant growth promoter properties, extracted from seeds, cotyledons or plantlets of the genus Lupinus, and to its application in the control of pathogenic agents that attack plants and as a plant bio-stimulant. This polypeptide may be applied directly on plants, or the plants may be genetically modified to express the polypeptide in their tissues. In addition, due to its unusual inherent characteristics, the polypeptide may be utilized in the preparation of protein concentrates useful as supplements in the diet of man and other animals.
The present invention also describes the nucleotide DNA sequence corresponding to the gene fragment that encodes the Lupinus polypeptide, as well as its sequence of amino acid residues, microorganisms transformed with the gene fragment that encodes the Lupinus polypeptide and methods for its application as a fungicide, insecticide, plant growth promoter or fertilizer or as a supplement in human or animal nutrition.
It is also an object of this invention the polypeptide characterized by the sequence of amino acid residues referred above, in which one or more amino acid residues are absent, have been substituted or added, or maintaining its biological activities after suffering chemical modification, such as, for example, glycosylation.
The control of pathogenic agents constitutes a serious problem worldwide with respect to the most important crops. Pathogenic fungi are particularly important in what concerns the storage of agricultural products. Presently, control over fungal growth is generally achieved by massive applications of chemical fungicides. However, phytopharmaceutical products currently available in the market display several serious disadvantages. On one hand, they exhibit high economical and environmental costs; on the other hand, many fungal species have been developing resistance mechanisms to some top fungicides, often turning them obsolete in a couple of years after their introduction in the market.
Even though plants do not possess an immunological system resembling that of animals, they have evolved an inherent resistance to the attack of pathogenic fungi. However, the techniques employed for plant growth, harvest and storage in modern agriculture promote very often good or optimal conditions for pathogen development.
In addition, the number of microbial pathogens that may affect and harm plant crops is quite high. As an example, the following genera may be referred: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Gaeumanomyces, Macrophomina, Nectria, Phoma, Phomopsis, Phymatotrichum, Phytophthora, Plasmopara, Puccinia, Pythium, Rhizoctonia, Uncinula, and Verticillium. The application of the fungicides currently available in the market is limited to some of these genera, and is not an effective solution in the control of plant infections.
An alternative strategy in the fight against microbial pathogens is the identification and purification of substances of biological origin with potent antifungal activity. The identification of such compounds involves searching a variety of organisms, such as plants and microorganisms, for substances that are subsequently tested in antifungal assays and finally isolated and characterized.
In this way, many classes of antifungal proteins have already been isolated, including chitinases, cystein-rich proteins that bind strongly to chitin, β-1,3-glucanases, permeatins, thionins and lipid transfer proteins. These proteins are thought to play a fundamental role in the natural defenses of plants against the attack of pathogens.
Several methodologies are described in the available literature on the utilization of antifungal proteins, extracted from plants or microorganisms, either for direct application over the pathogenic agents, or in transgenic plants expressing those proteins. The antifungal proteins most often utilized in these methodologies include chitinases, glucanases, osmotin-type proteins and lysozymes. Various studies have demonstrated that genetically modified plants over-expressing these proteins exhibit enhanced resistance to many pathogens (EP 0392 225).
Modern techniques of Molecular Biology allowed the development of recombinant DNA technology and, consequently, plant transformation with genes encoding antifungal proteins. This procedure usually involves insertion of the gene encoding the protein of interest in a plant tissue, followed by regeneration of a whole plant from the genetically modified plant tissue.
However, the activity of some of these proteins is reduced by the presence of ions, in particular potassium, sodium or calcium. For this reason, although the proteins may exhibit a potent antifungal activity in in vitro assays, they may be ineffective in vivo due to the high physiological concentrations of the ions that may naturally occur in the transformed plant tissues.
Document “Ramos, Paula Cristina Rodrigues, et. al—“Accumulation of a lectin-like breakdown product of beta-conglutin catabolism in cotyledons of germinating Lupinus albus L. seeds” Planta (Heidelberg), vol. 203, n° 1, 1997, pages 26-34″ describes isolation and purification of a 20-kDa polypeptide which is an intermediate breakdown product of beta-conglutin catabolism, the vicilin-like storage protein, which interacts with a variety of glycoproteins and possesses a lectin-type activity. In Table 1 of this document, several N-terminal amino acid sequences are described including the one of the 20-kDa polypeptide isolated from L. albus seeds. However, the N-terminal sequence presented in the Table 1 of this document includes exclusively the identity of residues no. 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 20, and 23. This corresponds to a total of 15 residues out of the 173 amino acid residues that compose SEQ ID NO: 1 of the present invention. In fact, the N-terminal sequence of this document is not the responsible for the biological properties related to the polypeptide of the present invention. Moreover, the N-terminal sequence described in the referred document is intended to be used to construct suitable primers, which may then be utilized in molecular biology as the first step of the long and uncertain process of gene and protein sequencing, while the main purpose of the present invention is to disclose a polypeptide exhibiting several biological activities.
The document “Database UniProt [Online] 16 Aug. 2004 (2004 Aug. 16)—“Beta-conglutin” discloses a protein obtained from Lupinus albus, namely beta-conglutin. This protein is the major seed storage protein present in Lupinus seeds. Despite being the precursor of the polypeptide with the sequence of SEQ ID NO:1, according to the present invention, these two polymers correspond to distinct molecules for a number of reasons, namely a) beta-conglutin is regarded as a protein, with a typical quaternary structure, whereas the molecule with the sequence of SEQ ID NO:1 should not be considered as a protein, but rather as a polypeptide; b) beta-conglutin, much like the vast majority of proteins, is easily destroyed by harsh conditions. On the opposite, the polypeptide of the present invention is extremely resistant to denaturation, withstanding prolonged boiling, treatment with strong acids or bases, detergents and organic solvents. In addition, it is resistant to the Sun ultraviolet radiation; c) these two molecules fulfil entirely different roles in nature—whereas beta-conglutin is a seed storage protein, the polypeptide of the present invention displays a protective role in the plant, exhibiting a potent antifungal activity and a growth promoter activity. On the other hand, whereas beta-conglutin possesses strong catalytic activities of chitosanase and chitinase, the polypeptide of the present invention exhibits catalytic activity of beta-N-acetyl-D-glucosaminidase and a low level of chitosanase activity.
Document “Database UniProt [Online] 24 May 2005 (2005 May 24)—“Vicilin-like protein (fragment)” discloses a fragment (length: 531 amino acid residues; molecular mass: 62,032 Da) of the same protein (i.e. beta-conglutin) described in the previous document (length: 533 amino acid residues; molecular mass: 62,130 Da). Therefore, all remarks made above in what concerns this document apply equally to the document under analysis.
In conclusion, it is becoming imperative the identification and purification of novel compounds of biological origin exhibiting anti-pathogen properties in the fight against the pathogens that affect plants. Particular importance must be given to those compounds that are effective over a wide range of pathogens and that maintain the biological activity under in vivo conditions.
Agricultural practices have been optimized, over a long time period, to promote plant growth and development and to increase crop production. On the other hand, it is predictable that in the medium to long term, food shortage may occur in many areas of the globe. Current techniques to control plant growth under environmentally controlled conditions are expensive and require complex equipment. For these reasons, many researchers have searched for and have reported physiologically active substances, either natural or synthetic, that exhibit a boosting effect upon the growth and development of crops. However, only a few of these substances have found practical application under real, agricultural conditions. Therefore, it is also increasingly important to discover or to develop plant growth promoters that are environmentally friend and that present no toxicity to man, animals and the environment.
Plant legumes or, more specifically, their seeds, are considered as the major source of protein worldwide for animal and human nutrition. In this respect, soybean fulfills a prominent role, not only for the high protein content and quality of its seeds, but also for their richness in oil. However, from the agricultural point of view, soybean requires fertile soils and an abundant water supply. Plants belonging to the Lupinus genus have conquered, over the last few decades, a relevant, strong and of great potential position in comparison to soybean. If, on one hand, their seeds possess protein and oil levels comparable to those of soybean, on the other, their species are well adapted to poor soils and to conditions of low water availability. For these reasons, lupins have sometimes been considered as the “poor cousins” of soybean.
The high level of alkaloids which are toxic to animals and which are naturally present in traditional, wild-type lupin seeds have long hampered the generalized cultivation of Lupinus species and the use of their seeds for animal and human consumption. This is the main reason why lupin cultivation has lagged far behind that of soybean. In Portugal, for example, traditional consumption of lupin seeds has long been associated to beer ingestion. These seeds are first boiled in water (heating at 100° C. destroys the capacity of seeds to germinate but does not block the imbibition's process) and then immersed under running water for a few days to remove the toxic alkaloids. However, the recent application of breeding techniques allowed the development of the so called sweet lupin varieties, characterized by containing a low seed alkaloid content (<0.004% w/w), as opposed to the traditional bitter cultivars (alkaloid content >0.004% w/w). For this reason, the seeds of sweet lupin varieties may be safely utilized as human and animal feed.
It is therefore predictable an increasing development of lupin-derived food products for both human and animal nutrition, as has happened a number of decades ago with soybean. This is particularly important in the case of lupin seed proteins, either albumins or globulins.